Model animal for pregnancy-induced hypertension syndrome, and treatment method therefor

ABSTRACT

A lentiviral vector was used to produce non-human animals that express human sFLT1 specifically in the murine placenta, to provide model animals of diseases such as pregnancy-induced hypertension syndrome that are close to the clinical conditions, methods for producing the model animals, methods of screening for candidate compounds as therapeutic agents for diseases such as pregnancy-induced hypertension syndrome by using the model animals, and therapeutic agents for diseases such as pregnancy-induced hypertension syndrome. As a result, the model animals were found to exhibit symptoms that are very close to the clinical conditions in human, which are presentation of hypertension as well as placental insufficiency, intrauterine growth retardation, glomerulosclerosis, and proteinuria during pregnancy, and improvement of those symptoms postpartum. Furthermore, when pravastatin was administered to this model animal, it was found that diseases such as pregnancy-induced hypertension syndrome were improved by the activation of placenta-derived growth factor (PIGF) which antagonizes sFLT1.

TECHNICAL FIELD

The present invention relates to model animals for diseases such aspregnancy-induced hypertension syndrome that shows placenta-specificexpression of sFLT1, and methods for producing the model animals.Furthermore, the present invention relates to methods of screening forcandidate compounds as therapeutic agents for diseases such aspregnancy-induced hypertension syndrome and pharmaceutical compositionsfor treating diseases such as pregnancy-induced hypertension syndrome.

BACKGROUND ART

Pregnancy-induced hypertension syndrome arises from placentalinsufficiency, and is observed in approximately 5 to 7% of pregnantwomen (Lancet. 2006 Apr. 1; 367(9516): 1066-74, WHO analysis of causesof maternal death: a systematic review (Non-patent Document 1)).Pregnancy-induced hypertension syndrome is a major cause of maternal andinfant morbidity and mortality, and the only established therapy is toterminate pregnancy and remove the placenta. Therefore, generating asuitable animal model is extremely significant clinically forunderstanding the cause of the disease and developing therapeuticagents. Maynard et al. have reported that an increase of soluble FLT1(sFLT1) and decreases of VEGF and PIGF (placental growth factor,placenta-derived VEGF-like protein) in the mother's blood are observedin women with pregnancy-induced hypertension syndrome (J Clin Invest.2003 March; 111(5): 649-58, Excess placental soluble fms-like tyrosinekinase 1 (sFlt1) may contribute to endothelial dysfunction,hypertension, and proteinuria in preeclampsia. (Non-patent Document 2)).Since sFLT1 is a soluble form of a VEGF receptor, and antagonizes thefunctions of VEGF and PIGF, it is considered that sFLT1 causesfunctional disorders of the vascular endothelium and pregnancy-inducedhypertension syndrome due to the impairment of angiogenic signaling (NEngl J Med. 2004 Feb. 12; 350(7): 672-83. Circulating angiogenic factorsand the risk of preeclampsia. (Non-patent Document 3)). This is alsosupported by the fact that systemic administration of an adenovirusvector (AdV−) that expresses sFLT1 to pregnant rats showed hypertension,proteinuria, and glomerulosclerosis which are classical pathologicalchanges in pregnancy-induced hypertension syndrome (J Clin Invest. 2003March; 111(5): 649-58, Excess placental soluble fms-like tyrosine kinase1 (sFlt1) may contribute to endothelial dysfunction, hypertension, andproteinuria in preeclampsia. Long-term maternal cardiovascular functionin a mouse model of sFlt-1-induced preeclampsia. (Non-patent Document2)). However, while sFLT1 is expressed in pregnancy-induced hypertensionsyndrome until delivery, sFLT1 expression in a model animal wastransient. Furthermore, in conventional model animals, since sFLT1 isproduced in the mother's body (mainly liver) but not in the placentawhich is the causative organ, the pathological condition does notimprove upon delivery. That is, the conventional model animals aredefective as models for pregnancy-induced hypertension syndrome whichshows improvement of pathological conditions upon delivery.

Meanwhile, it has been proven that when blastocyst-stage embryos areinfected with a lentiviral vector, genes can be introduced into theplacenta alone without being transfected into the mother's body and theunborn baby (Non-patent Documents 4 and 5).

PRIOR ART DOCUMENTS Patent Documents

-   -   [Patent Document 1] WO 2007/020786

Non-Patent Documents

-   -   [Non-patent Document 1] Lancet. 2006 April 1; 367(9516):        1066-74, WHO analysis of causes of maternal death: a systematic        review    -   [Non-patent Document 2] J Clin Invest. 2003 March; 111(5):        649-58, Excess placental soluble fms-like tyrosine kinase 1        (sFlt1) may contribute to endothelial dysfunction, hypertension,        and proteinuria in preeclampsia.    -   [Non-patent Document 3] N Engl J Med. 2004 Febuary 12; 350(7):        672-83. Epub 2004 Feb. 5, Circulating angiogenic factors and the        risk of preeclampsia.    -   [Non-patent Document 4] Nat Biotechnol. 2007 February; 25(2):        233-7. Complementation of placental defects and embryonic        lethality by trophoblast-specific lentiviral gene transfer.    -   [Non-patent Document 5] Genesis. 2009 December; 47(12): 793-8.,        Placenta-specific gene activation and inactivation using        integrase-defective lentiviral vectors with the Cre/LoxP system.

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

The present invention was achieved in view of the above circumstances.An objective of the present invention is to provide model animals fordiseases such as pregnancy-induced hypertension syndrome which are closeto the clinical conditions, and methods for producing the model animals.Another objective of the present invention is to provide methods ofscreening for candidate compounds as therapeutic agents for diseasessuch as pregnancy-induced hypertension syndrome, agents for treatingdiseases such as pregnancy-induced hypertension syndrome, and such usingthe model animals.

Means for Solving the Problems

To solve the above-mentioned problems, the present inventors aimed toproduce non-human animals that express human sFLT1 specifically in themouse placenta using lentiviral vectors. As a result, the presentinventors discovered that the model animals exhibit hypertension,placental insufficiency, intrauterine growth retardation (IUGR),glomerulosclerosis, and proteinuria during pregnancy, and improvement ofthose symptoms postpartum, which are symptoms very close to the clinicalconditions in human.

Furthermore, the present inventors administered pravastatin (PS) tothese model animals. As a result, the present inventors discovered thatpravastatin induces expression of placenta-derived growth factor (PIGF)which antagonizes sFLT1, and improves symptoms of pregnancy-inducedhypertension syndrome, placental insufficiency, intrauterine growthretardation, glomerulosclerosis, and proteinuria caused by sFLT1.Furthermore, the present inventors discovered that pravastatin inducesectopic expression of PIGF in sites other than the placenta.

The present invention is based on such findings, and relates to thefollowing invention:

-   [1] a non-human mammal that expresses sFLT1 specifically in the    placenta;-   [2] the non-human mammal of [1], which exhibits symptoms of at least    one disease selected from the group consisting of pregnancy-induced    hypertension syndrome, placental insufficiency, intrauterine growth    retardation, glomerulosclerosis, and proteinuria;-   [3] the non-human mammal of [2], which is obtained by the steps    of (a) to (c) below:    -   (a) removing the zona pellucida of a blastocyst of a non-human        mammal;    -   (b) introducing an sFLT1-encoding nucleic acid specifically into        the trophectoderm of the blastocyst obtained in step (a); and    -   (c) transplanting the blastocyst obtained in step (b) into a        recipient;-   [4] a method of producing a non-human mammal that comprises the    steps of (a) to (c) below:    -   (a) removing the zona pellucida of a blastocyst of a non-human        mammal;    -   (b) introducing an sFLT1-encoding nucleic acid specifically into        the trophectoderm of the blastocyst obtained in step (a); and    -   (c) transplanting the blastocyst obtained in step (b) into a        recipient;-   [5] a method of screening for a substance that improves the symptoms    of pregnancy-induced hypertension syndrome, placental insufficiency,    intrauterine growth retardation, glomerulosclerosis, and    proteinuria, which comprises the steps of (a) to (c) below:    -   (a) obtaining the non-human mammal of any one of [1] to [3];    -   (b) administering a test substance to the non-human mammal        obtained in step (a); and    -   (c) selecting a substance that improves at least one symptom of        pregnancy-induced hypertension syndrome, placental        insufficiency, intrauterine growth retardation,        glomerulosclerosis, and proteinuria, as compared to when the        test substance is not administered;-   [6] a pharmaceutical composition for use in treating at least one    disease selected from the group consisting of pregnancy-induced    hypertension syndrome, placental insufficiency, intrauterine growth    retardation, glomerulosclerosis, and proteinuria, which comprises a    statin as an active ingredient;-   [7] a pharmaceutical composition for use in treating at least one    disease selected from the group consisting of pregnancy-induced    hypertension syndrome, placental insufficiency, intrauterine growth    retardation, glomerulosclerosis, and proteinuria, which comprises    PIGF or a PIGF-encoding nucleic acid as an active ingredient;-   [8] a method for treating at least one disease selected from the    group consisting of pregnancy-induced hypertension syndrome,    placental insufficiency, intrauterine growth retardation,    glomerulosclerosis, and proteinuria, which comprises the step of    administering a statin to a subject;-   [9] a method for treating at least one disease selected from the    group consisting of pregnancy-induced hypertension syndrome,    placental insufficiency, intrauterine growth retardation,    glomerulosclerosis, and proteinuria, which comprises the step of    administering PIGF or a PIGF-encoding nucleic acid to a subject;-   [10] use of a statin in the manufacture of a pharmaceutical    composition used for treating at least one disease selected from the    group consisting of pregnancy-induced hypertension syndrome,    placental insufficiency, intrauterine growth retardation,    glomerulosclerosis, and proteinuria;-   [11] use of PIGF or a PIGF-encoding nucleic acid in the manufacture    of a pharmaceutical composition used for treating at least one    disease selected from the group consisting of pregnancy-induced    hypertension syndrome, placental insufficiency, intrauterine growth    retardation, glomerulosclerosis, and proteinuria;-   [12] a statin for use in treating at least one disease selected from    the group consisting of pregnancy-induced hypertension syndrome,    placental insufficiency, intrauterine growth retardation,    glomerulosclerosis, and proteinuria;-   [13] PIGF or a PIGF-encoding nucleic acid for use in treating at    least one disease selected from the group consisting of    pregnancy-induced hypertension syndrome, placental insufficiency,    intrauterine growth retardation, glomerulosclerosis, and    proteinuria;-   [14] use of a statin for treating at least one disease selected from    the group consisting of pregnancy-induced hypertension syndrome,    placental insufficiency, intrauterine growth retardation,    glomerulosclerosis, and proteinuria;-   [15] use of PIGF or a PIGF-encoding nucleic acid for treating at    least one disease selected from the group consisting of    pregnancy-induced hypertension syndrome, placental insufficiency,    intrauterine growth retardation, glomerulosclerosis, and    proteinuria;-   [17] an agent for promoting PIGF induction comprising a statin as an    active ingredient;-   [18] a method for inducing PIGF, which comprises the step of    administering a statin to a subject;-   [19] use of a statin in the manufacture of a PIGF-inducing agent;-   [20] a statin for use in inducing PIGF; and-   [21] use of a statin for inducing PIGF.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows in diagrams and photographs a pregnancy-inducedhypertension syndrome model generated by expressing sFLT1 specificallyin the placenta. (a) shows a scheme for generating pregnancy-inducedhypertension syndrome model mice. After removing the zona pellucida,blastocysts were transduced with a lentiviral vector (LV-hsFLT1)expressing human sFLT1 (hsFLT1) and then transplanted intopseudo-pregnant female animals. The transduced trophectoderm (TE) celllineage provides the main components of the placenta and continuouslyexpresses hsFLT1. ICM: inner cell mass. (b) Photographs showing theresults obtained when genomic DNA (gDNA) and mRNA collected from theindicated tissues at E13.5 were subjected to PCR and RT-PCR,respectively. Actb was used as control. pl: placenta; fe: fetus; ut:uterus; and li: liver. (c-f) Graphs showing results of measuring thehsFLT1 concentration (c and d), blood pressure (e), and ratio of urinaryalbumin to creatinine (f) in the pregnant female animals. (c) Theconcentration of hsFLT1 circulating in the mother's blood on the 18thday of pregnancy (at E18.5) depended on the amount of LV-hsFLT1 used fortransduction. (d) hsFLT1 circulating in the mother's body increasedduring pregnancy. (e) Hypertension was observed at E16.5 and E18.5depending on the amount of LV-hsFLT1. The blood pressure became normalafter delivery of the placenta. PD: post-delivery. (f) The ratio ofurinary albumin to creatinine was significantly higher in the LV-hsFLTgroup than in the control LV-EGFP group (P<0.05).

FIG. 2 shows in graphs that pravastatin (PS) improves pregnancy-inducedhypertension syndrome by up-regulating PIGF. (a) Hypertension induced bythe placenta-specific hsFLT1 expression was improved by pravastatin.Pravastatin (5 μg) was intraperitoneally administered to the femaleanimals every day starting at the indicated days. (b) The concentrationsof hsFLT1, mouse VEGF (mVEGF), and mouse PIGF (mPIGF) in the mother'sblood were measured at E18.5. (c) mRNA extracted from the placenta atE18.5 was analyzed by quantitative RT-PCR. (d) Pravastatin (5 μg) wasadministered every day starting at E7.5 to non-pregnant and pregnantfemale mice that had not been treated with viral vector. The mPIGFconcentration in the mother's blood was measured at E18.5. (e) HUVECcells were cultured in a statin-containing medium, and 24 hours later,the concentrations of human VEGF (hVEGF) and human PIGF (hPIGF) in thesupernatant were measured. 293T cells were used as a control.

FIG. 3 shows in photographs and graphs that the pravastatin or LV-mPIGFtreatment restored placental formation and ameliorated IUGR inpregnancy-induced hypertension syndrome. (a-d) Placenta-specific hsFLT1overexpression impaired vasculogenesis in the placenta and caused IUGR.(a) Impaired vasculogenesis in the placenta was restored by pravastatintreatment or PIGF expression. Placentas were collected at E13.5, andPECAM1-expressing cells were stained with an anti-CD31 antibody. Thewhite box in the upper panel is magnified in the lower panel. (b-d)Fetuses and placentas were collected by Caesarian section at E18.5. (b)Live pups. (c) Placentas. Photos were taken from the maternal side andfetal side (lower part). (d) Fetal weight and placental weight.

FIG. 4 shows in photographs the HE staining result of kidney sectionscollected from a female on the 18th day of pregnancy. The treatment isindicated in the upper left of each photograph. As compared to thecontrol (upper left), shrinking of the kidney glomeruli was observed dueto placenta-specific hsFLT1 overexpression (upper right). The pathologywas recovered by administering pravastatin (lower left) oroverexpressing mPIGF specifically in the placenta (lower right) from theseventh day of pregnancy.

FIG. 5 shows in a graph changes in the blood pressure due to pravastatin(PS) administration or placenta-specific mPIGF overexpression. The bloodpressure did not change even when pravastatin was administered towild-type pregnant mice from the seventh day to the 18th day ofpregnancy (solid line). Furthermore, even when mPIGF was overexpressedspecifically in the placenta, the blood pressure did not change (dottedline).

MODE FOR CARRYING OUT THE INVENTION

The present invention relates to non-human mammals that express solublefms-like tyrosine kinase-1 (sFLT1) specifically in the placenta. In apreferred embodiment, non-human mammals of the present inventionconstitutively express sFLT1. In non-human mammals of the presentinvention, sFLT1 expression is placenta-specific, but placenta-derivedsFLT1 circulates in the mother's blood. In the placenta, blood vesselsderived from the mother's body and fetus form a very intricate structure(labyrinthine layer) where the blood flows of the mother and the fetusdo not mix but gases, hormones, nutrients, and waste are exchanged.Fetus-derived cells are also known to infiltrate into some of themother's blood vessels. Since sFLT1 produced by fetus-derived cells ofthe placenta is secreted to the outside of the cells, this is collectedthrough the mother's blood vessels in the placenta, and circulates inthe mother's blood via the umbilical cord.

Non-human mammals of the present invention show symptoms of diseasessuch as pregnancy-induced hypertension syndrome, placentalinsufficiency, intrauterine growth retardation, glomerulosclerosis, andproteinuria. Furthermore, while sFLT1 expression is placenta-specific,placenta-derived sFLT1 circulates in the mother's blood. In addition,these mammals follow a course very similar to human pathology, such asshowing improvement of symptoms of these diseases after delivery. Thatis, in preferred embodiments, when the non-human mammals of the presentinvention have placentas, they exhibit symptoms of diseases such aspregnancy-induced hypertension syndrome, placental insufficiency,intrauterine growth retardation, glomerulosclerosis, and proteinuria.When the placenta is taken out from the body at delivery, theydemonstrate improvement of symptoms. Such non-human mammals of thepresent invention are useful as model animals for diseases such aspregnancy-induced hypertension syndrome, placental insufficiency,intrauterine growth retardation, glomerulosclerosis, and proteinuria.Furthermore, non-human mammals of the present invention can be used inmethods of screening for candidate compounds as pharmaceutical agentsfor treating or preventing diseases such as pregnancy-inducedhypertension syndrome, placental insufficiency, intrauterine growthretardation, glomerulosclerosis, and proteinuria.

In a preferred embodiment of the present invention, diseases such aspregnancy-induced hypertension syndrome, placental insufficiency,intrauterine growth retardation, glomerulosclerosis, and proteinuria arecaused by sFLT1.

FLT1 (membrane-type fms-like tyrosine kinase 1) is a membrane proteinknown as a specific receptor for vascular endothelial growth factor(VEGF) and placental growth factor (PIGF). On the other hand, sFLT1(soluble fms-like tyrosine kinase 1) is an FLT1 that does not have atransmembrane domain, and is a soluble protein known as a specificreceptor for vascular endothelial growth factor (VEGF) and placentalgrowth factor (PIGF). sFLT1 of the present invention is not limited aslong as the whole or a portion of the transmembrane domain is removedfrom full-length FLT1, and it is soluble and can dimerize. Thetransmembrane domain can be removed, for example, by protein cleavage,mRNA cleavage, or selective use of exons, without being limited thereto.

The nucleotide sequence and amino acid sequence of the transmembranedomain of human FLT1 identified using SOSUI are shown in SEQ ID NOs: 19and 20, and the nucleotide sequence and amino acid sequence of thetransmembrane domain of mouse FLT1 are shown in SEQ ID NOs: 21 and 22,but the sequences of the transmembrane domain are not limited thereto.

As described, those skilled in the art can easily identify thetransmembrane domain in FLT1 derived from various animal species byusing a well-known program such as SOSUI.

Furthermore, those skilled in the art can identify the transmembranedomain based on known publications. For example, extracellularly Flt-1has seven immunoglobulin-like domains: first to seventh; and sFlt-1 isknown to have the first to sixth domains among these seven domains, aswell as 31 amino acids encoded by the 5′ region of intron 13 (Shibuya,Cell Structure and Function, 26, 25-35 (2001)). The 31 amino acidsencoded by the 5′ region of intron 13 are known to be highly conservedamong mammals. Furthermore, the transmembrane domain is encoded by exon16 of the FLT gene. In fact, it is known that when exon 16 of human FLT1is deleted, it is converted to a soluble form (Molecular and CellularBiology, January 2005, p. 346-354, Vol. 25, No. 1, Membrane Fixation ofVascular Endothelial Growth Factor Receptor 1 Ligand-Binding Domain IsImportant for Vasculogenesis and Angiogenesis in Mice).

Those skilled in the art can obtain sFLT1 from humans, mice, or othermammals based on such information.

Without being limited thereto, examples of human sFLT1-encoding nucleicacids include the following sequences:

-   (1) a DNA having the nucleotide sequence (the nucleotide sequence of    SEQ ID NO: 1) specified by GenBank: U01134.1; and-   (2) a DNA having a nucleotide sequence in which a nucleotide    sequence of the transmembrane domain is removed from the nucleotide    sequence specified by NCBI Reference Sequence: NM_002019.4.

Furthermore, human sFLT1 proteins include the following sequences butare not limited thereto:

-   (3) proteins having amino acid sequences encoded by the DNAs of (1)    and (2) mentioned above;-   (4) a protein having the amino acid sequence (the amino acid    sequence of SEQ ID NO: 2) specified by GenBank: U01134.1 and    GenBank: AAC50060.1; and-   (5) a protein having an amino acid sequence in which an amino acid    sequence of the transmembrane domain is removed from the amino acid    sequence specified by NCBI Reference Sequence: NP_002010.2.

Furthermore, sFLT1 of the present invention is not limited tohuman-derived sFLT1. sFLT1 of the present invention include sFLT1derived from chimpanzees, mice, rats, dogs, cows, horses, pigs, andsheep. These animal-derived sFLT1 also have structures in which thetransmembrane domain is removed from full-length FLT1.

Examples of nucleic acids that encode the chimpanzee-derived sFLT1include nucleic acids having a nucleotide sequence in which a nucleotidesequence of the transmembrane domain is removed from the nucleotidesequence specified by NCBI Reference Sequence: XM_509605.2, but are notlimited thereto. Furthermore, examples of chimpanzee-derived sFLT1proteins include proteins having an amino acid sequence encoded by thenucleic acid mentioned above, and proteins having an amino acid sequencein which an amino acid sequence of the transmembrane domain is removedfrom the amino acid sequence specified by NCBI Reference SequenceXP_509605.2, but are not limited thereto.

Examples of nucleic acids that encode the mouse-derived sFLT1 include anucleic acid having the nucleotide sequence specified by GenBank:BC029674.1, and nucleic acids having a nucleotide sequence in which anucleotide sequence of the transmembrane domain is removed from thenucleotide sequence specified by NCBI Reference Sequence: NM_010228.3,but are not limited thereto. Furthermore, examples of mouse-derivedsFLT1 proteins include proteins having an amino acid sequence encoded bythe nucleic acid mentioned above, and proteins having an amino acidsequence in which an amino acid sequence of the transmembrane domain isremoved from the amino acid sequence specified by NCBI ReferenceSequence NP_034358.2, but are not limited thereto.

Examples of nucleic acids that encode the rat-derived sFLT1 includenucleic acids having a nucleotide sequence in which a nucleotidesequence of the transmembrane domain is removed from the nucleotidesequence specified by NCBI Reference Sequence: NM_019306.1, but are notlimited thereto. Furthermore, examples of rat-derived sFLT1 proteinsinclude proteins having an amino acid sequence encoded by the nucleicacid mentioned above, and proteins having an amino acid sequence inwhich an amino acid sequence of the transmembrane domain is removed fromthe amino acid sequence specified by NCBI Reference SequenceNP_062179.1, but are not limited thereto.

Examples of nucleic acids that encode the dog-derived sFLT1 includenucleic acids having a nucleotide sequence in which a nucleotidesequence of the transmembrane domain is removed from the nucleotidesequence specified by NCBI Reference Sequence: XM_534520.2, but are notlimited thereto. Furthermore, examples of dog-derived sFLT1 proteinsinclude proteins having an amino acid sequence encoded by the nucleicacid mentioned above, and proteins having an amino acid sequence inwhich an amino acid sequence of the transmembrane domain is removed fromthe amino acid sequence specified by NCBI Reference SequenceXP_534520.2, but are not limited thereto.

Examples of nucleic acids that encode the cow-derived sFLT1 includenucleic acids having a nucleotide sequence in which a nucleotidesequence of the transmembrane domain is removed from the nucleotidesequence specified by NCBI Reference Sequence: XM_001249768.2, but arenot limited thereto. Furthermore, examples of cow-derived sFLT1 proteinsinclude proteins having an amino acid sequence encoded by the nucleicacid mentioned above, and proteins having an amino acid sequence inwhich an amino acid sequence of the transmembrane domain is removed fromthe amino acid sequence specified by NCBI Reference SequenceXP_001249768.2, but are not limited thereto.

Examples of nucleic acids that encode the horse-derived sFLT1 includenucleic acids having a nucleotide sequence in which a transmembranedomain nucleotide sequence is removed from the nucleotide sequencespecified by NCBI Reference Sequence: XM_001492325.2, but are notlimited thereto. Furthermore, examples of horse-derived sFLT1 proteinsinclude proteins having an amino acid sequence encoded by the nucleicacid mentioned above, and proteins having an amino acid sequence inwhich an amino acid sequence of the transmembrane domain is removed fromthe amino acid sequence specified by NCBI Reference SequenceXP_001492375.1, but are not limited thereto.

Examples of nucleic acids that encode the pig-derived sFLT1 includenucleic acids having a nucleotide sequence in which a nucleotidesequence of the transmembrane domain is removed from the nucleotidesequence specified by NCBI Reference Sequence: XM_001925740.1, but arenot limited thereto. Furthermore, examples of pig-derived sFLT1 proteinsinclude proteins having an amino acid sequence encoded by the nucleicacid mentioned above, and proteins having an amino acid sequence inwhich an amino acid sequence of the transmembrane domain is removed fromthe amino acid sequence specified by NCBI Reference Sequence XP_(—)001925775.1, but are not limited thereto.

Examples of nucleic acids that encode the sheep-derived sFLT1 includenucleic acids having a nucleotide sequence in which a nucleotidesequence of the transmembrane domain is removed from the nucleotidesequence specified by GenBank: AF233077.1, but are not limited thereto.Furthermore, examples of sheep-derived sFLT1 proteins include proteinshaving an amino acid sequence encoded by the nucleic acid mentionedabove, and proteins having an amino acid sequence in which an amino acidsequence of the transmembrane domain is removed from the amino acidsequence specified by GenBank: AAF60281.1, but are not limited thereto.

In the present invention, “nucleic acids encoding sFLT1” include“sFLT1-encoding DNAs” and “sFLT1-encoding RNAs”.

In the present invention, “pregnancy-induced hypertension syndrome”refers to observation of either hypertension or hypertension accompaniedby proteinuria after the 20th week of pregnancy and until 12 weekspostpartum, and these symptoms are not simply due to incidentalcomplications of pregnancy. Progression of pregnancy-inducedhypertension syndrome leads to placental insufficiency, intrauterinegrowth retardation, glomerulosclerosis, and proteinuria.

A non-human mammal of the present invention that shows placenta-specificexpression of sFLT1 can be produced by the steps of (a) to (c) below:

-   (a) removing the zona pellucida of a blastocyst of a non-human    mammal;-   (b) introducing an sFLT1-encoding nucleic acid specifically into the    trophectoderm of the blastocyst obtained in step (a); and-   (c) transplanting the blastocyst obtained in step (b) into a    recipient.

In the present invention, the recipient is preferably a non-human mammal

Non-human animals obtained by such steps exhibit symptoms of diseasessuch as pregnancy-induced hypertension syndrome, placentalinsufficiency, intrauterine growth retardation, glomerulosclerosis, andproteinuria.

First, in the production of non-human mammals of the present invention,blastocysts are obtained. A blastocyst refers to an embryo that hascompleted the cleavage stage in the early development of mammals.Mammalian eggs are alecithal; and they divide holoblastically and formaggregates of blastomeres. At the 32-cell stage, eggs divide into innercell mass inside the aggregate and trophectoderm which enfolds theoutside of the aggregate. The inner cell mass develops into the body ofa fetus in the future, while the trophectoderm differentiates into theplacenta in the future. In the methods of the present invention,sFLT1-encoding nucleic acids are introduced specifically intotrophectodermal cells that form the outermost layer of a blastocyst.

Animals from which the blastocysts are derived include non-humananimals. Examples of non-human animals include humans, mice, rats,rabbits, guinea pigs, hamsters, dogs, cats, cows, horses, pigs, goats,and sheep, but are not particularly limited thereto.

Blastocysts can be prepared by the methods described below. For example,eggs and sperms are collected from any non-human mammals, and thenfertilization is carried out by methods known to those skilled in theart. Blastocysts can be prepared from the resulting fertilized eggs bymethods known to those skilled in the art, for example, by culturingeggs in a kSOM medium for 96 hours. Alternatively, blastocysts may beobtained directly from animals according to methods that areconventionally used by those skilled in the art (for example, methodsdescribed in: Manipulating the mouse embryo, a laboratory manual, 3rdedition, p 201-203, Cold Spring Harbor Laboratory Press).

In the non-human mammal production of the present invention, the zonapellucida is then removed from the blastocysts obtained above. Embryos(preimplantation early embryos) are covered with zona pellucida, whichis an extracellular matrix, to protect them from infection of viruses orthe like. In the present invention, the zona pellucida is removed.

The zona pellucida can be removed by known methods, for example, acidtreatment, enzyme treatment, or physical treatment. One example of suchacid treatment is a treatment that uses acidic Tyrode's solution(Manipulating the mouse embryo, a laboratory manual, 3rd edition, p485-486, Cold Spring Harbor Laboratory Press). The zona pellucida ispartially dissolved, for example, by sucking acidic Tyrode's solution(pH 2.3 to 2.5) with a micropipette and then gently spraying immobilizedembryos with the solution. Alternatively, the zona pellucida isdissolved by immersing embryos in acidic Tyrode's solution.

Enzyme treatment for removal of zona pellucida includes Pronasetreatment (Calbiochem 537088, Sigma P5147) and the like. For example,embryos are placed in a solution of 0.5% Pronase until the zonapellucida dissolves. After the zona pellucida dissolves, the embryos arewashed well with a medium, and then zona pellucida-removed blastocystscan be obtained (Manipulating the mouse embryo, a laboratory manual, 3rdedition, p 731, Cold Spring Harbor Laboratory Press).

Furthermore, in the present invention, zona pellucida may also beremoved by physical methods. Such physical methods of removal include azona pellucida dissection method in which embryos are immobilized andpart of the zona pellucida is dissected with a micropipette (Hum.Reprod., 5: 7-13, 1990), methods for dissecting, drilling, or thinningzona pellucida with a laser (Hum. Reprod., 15: 1061-1064, 2000) orpiezomicromanipulator (Developmental Biology, 250: 348-357, 2002) andthe like.

Zona pellucida can be removed by the methods described above. In thepresent invention, however, methods for removing zona pellucida are notlimited to the examples described above, and the methods include allmethods that can remove zona pellucida.

In the non-human mammal production of the present invention,sFLT1-encoding nucleic acids are then introduced into zonapellucida-removed blastocysts. In a preferred embodiment, methods forintroducing sFLT1-encoding nucleic acids into zona pellucida-removedblastocysts include methods for introducing a vector carrying ansFLT1-encoding nucleic acid into blastocysts, but are not particularlylimited thereto.

Vectors carrying an sFLT1-encoding nucleic acid to be introduced intoblastocysts include viral vectors. Viral vectors include DNA viralvectors and RNA viral vectors. DNA viral vectors include adenovirusvectors, adeno-associated virus vectors, and such, but are not limitedthereto. Furthermore, RNA viral vectors include retroviral vectors(Molecular Therapy 2003, 8; 666-673) such as lentiviral vectors(Molecular Therapy 2003, 8: 666-673). Furthermore, as described later,reagents for introducing nucleic acids such as HVJ liposomes,Lipofectamine, and such can also be used. HVJ (hemagglutinating virus ofJapan: Sendai virus)-liposome is a vector that can efficiently introduceliposome-encapsulated genes and oligonucleotides into various organs ina living body, by using the DNA-binding protein HMG-1 and the activityof a fusion protein of HVJ which is a virus that causes cell fusion.

Of such vectors, lentiviral vectors are preferred. Lentivirus belongs tothe retrovirus family, and is an immunodeficiency virus in human,monkey, cat, and bovine. With respect to gene structure, the virus isconstituted by several regulatory genes in addition to structural genesfundamental to the retrovirus (gag, pol, and env). Lentiviral vectorsconstructed by altering the lentivirus can integrate foreign genes intochromosome, and thus long term expression of the genes introducedtherein can be expected. Furthermore, unlike other retroviral vectors,lentiviral vectors have a nuclear translocation signal, and thus canintroduce genes into non-dividing cells.

Lentiviral vectors used in the present invention include all lentiviralvectors having at least LTR, RRE, and GAG Lentiviral vectors used in thenon-human mammal production of the present invention should have atleast these requirements, but may additionally have other genes, forexample, deltaU3, PPT, and WPRE. Such lentiviral vectors are alsopreferably used as the viral vector in the methods of the presentinvention.

In a particularly preferred embodiment, lentiviral vectors used in thepresent invention have LTR (deltaU3), GAG, RRE, PPT, and WPRE. Arepresentative example of lentiviral vectors having such structures is avector constructed by substituting a cDNA of interest for the GFP moietyof the GFP viral vector disclosed in the following document: MolecularTherapy 2003, 8: 666-673. The vector structure and construction methodare disclosed in the document indicated above.

In the present invention, the timing and amount of the sFLT1-encodingnucleic acid introduced into blastocysts are not particularly limited.However, preferably it is introduced at a quantity and timing that leadsto presentation of symptoms of pregnancy-induced hypertension syndromein a recipient transplanted with the sFLT1-introduced blastocysts. Thedose is preferably 10 to 1,000 ng-p24/mL, more preferably 20 to 500ng-p24/mL, and even more preferably 100 to 500 ng-p24/mL, but is notlimited thereto. Furthermore, the timing of introducing thesFLT1-encoding nucleic acid into blastocysts is preferably beforeimplantation.

Blastocysts can be infected with a lentiviral vector carrying ansFLT1-encoding nucleic acid, for example, by mixing zonapellucida-removed blastocysts with a solution containing the lentiviralvector carrying the sFLT1-encoding nucleic acid, and then leaving themixture to stand for 4 to 5 hours. By this method, a lentiviral vectorcarrying an sFLT1-encoding nucleic acid can be specifically introducedinto the trophectoderm.

Alternatively, sFLT1-encoding nucleic acids may also be introduced intozona pellucida-removed blastocysts by using a nucleic acid transfectionreagent. Herein, the nucleic acid transfection reagent refers to anyreagent that can introduce an sFLT1-encoding nucleic acid intoblastocysts. Such nucleic acid transfection reagents include, but arenot limited to, for example, Lipofectoamine 2000 (Invitrogen), Effectene(Qiagen), and FuGene (Roche).

Herein, an sFLT1-encoding nucleic acid includes sFLT1-encoding DNA andsFLT1-encoding RNA, but is not limited thereto. An sFLT1-encodingnucleic acid to be introduced may be in a form such as DNA, RNA, cDNA,mRNA, or artificial nucleic acid. Such DNAs, RNAs, cDNAs, and mRNAs alsoinclude derivatives thereof. Such artificial nucleic acids include, butare not limited to, for example, DNAs, RNAs, cDNAs, mRNAs with modifiedsugar chain structures, or derivatives thereof. Furthermore,sFLT1-encoding nucleic acids to be introduced may be naked DNAs orpolynucleotides introduced into a vector. Those skilled in the art candesign and use appropriate vectors depending on the purpose. Vectorsused in the present invention may comprise, in addition to ansFLT1-encoding nucleic acid to be introduced, polynucleotide regionsthat function in expression hosts, such as transcriptional initiationsite and transcription termination site, for more efficient expressionof the sFLT1-encoding nucleic acid.

Those skilled in the art can readily obtain sFLT1-encoding nucleicacids. For example, such polynucleotides can be isolated from naturalsources, using various biological samples such as placental tissues,trophoblast stem cells, and differentiated cells thereof as a source,based on their physicochemical properties and the like. Alternatively,the polynucleotides may be chemically synthesized based on knownsequence information.

sFLT1-encoding nucleic acids include homologous genes from variousanimals. Herein, “homologous gene” refers to the above-listedpolynucleotide comprising a nucleotide sequence of SEQ ID NO: 1, orpolynucleotides encoding a protein having a biological functionequivalent to that of the transcription/translation products of suchpolynucleotides, in various animals.

Methods that are well known to those skilled in the art for isolatinghomologous genes include hybridization techniques (Southern, E. M.,Journal of Molecular Biology, Vol. 98, 503, 1975) and polymerase chainreaction (PCR) techniques (Saiki, R. K., et al. Science, vol. 230,1350-1354, 1985; Saiki, R. K. et al. Science, vol. 239, 487-491, 1988).More specifically, those skilled in the art can routinely isolatesFLT1-encoding polynucleotides from various animal cells and tissues(for example, placental tissues, trophoblast stem cells, anddifferentiated cells thereof), using as a probe, polynucleotidesencoding sFLT1 (for example, the nucleotide sequence of SEQ ID NO: 1) ora portion thereof, or using as a primer, oligonucleotides thatspecifically hybridize to the sFLT1-encoding polynucleotides.Alternatively, sequences of homologous genes may be obtained from knowndatabases.

In order to isolate nucleic acids encoding such homologous genes, thehybridization reaction is usually performed under stringent conditions.Those skilled in the art can appropriately select stringenthybridization conditions. For example, hybridization may be performed bypre-hybridizing overnight at 42° C. in a hybridization solutioncontaining 25% formamide, or 50% formamide under more stringentconditions; 4×SSC; 50 mM Hepes, pH 7.0; 10×Denhardt's solution; and 20μg/mL denatured salmon sperm DNA, then adding a labeled probe, and thenincubating the solution overnight at 42° C. The subsequent washes can becarried out using a washing solution and temperature condition of “lxSSC, 0.1% SDS, 37° C.” or such, “0.5×SSC, 0.1% SDS, 42° C.” or such formore stringent conditions, or “0.2×SSC, 0.1% SDS, 65° C.” or such foreven more stringent conditions. With more stringent washing conditionsfor hybridization such as these, isolation of a DNA with higher homologyto the probe sequence can be expected. However, the above-mentionedcombinations of SSC, SDS, and temperature conditions are examples, andthose skilled in the art can suitably combine the above-mentionedfactors or with other factors (for example, probe concentration, probelength, hybridization reaction time, etc.) that determine hybridizationstringency to achieve similar stringency.

Isolation of DNAs with higher homologies can be expected underconditions of higher stringency such as conditions of 6 M urea, 0.4%SDS, and 0.1×SSC. High homology refers to sequence identities of atleast 50% or more, preferably 70% or more, more preferably 90% or more,and most preferably 95% or more of the whole amino acid sequence. Thenumber of amino acids to be altered in a mutant is generally 30 aminoacids or less, preferably 15 amino acids or less, more preferably 5amino acids or less, even more preferably 3 amino acids or less, and yeteven more preferably 2 amino acids or less.

The BLAST algorithm by Karlin and Altschul (Proc. Natl. Acad. Sci. USA87: 2264-2268, 1990; Proc. Natl. Acad. Sci. USA 90: 5873, 1993) can beused to determine the amino acid sequence identity and nucleotidesequence identity.

sFLT1-encoding polynucleotides of the present invention are notparticularly limited, but include preferably polynucleotides derivedfrom humans, mice, rats, rabbits, guinea pigs, hamsters, dogs, cats,cows, horses, pigs, goats, and sheep, and particularly preferablypolynucleotides derived from human.

In the methods of the present invention for producing non-human animals,the above blastocysts are finally transplanted into a recipient. In thepresent invention, the recipient is preferably a non-human animal. Therecipient is preferably the same animal or an animal belonging to thesame species as the animal from which the blastocysts are derived. Thoseskilled in the art can routinely transplant blastocysts into recipients(Manipulating the mouse embryo, a laboratory manual, 3rd edition, p263-271, Cold Spring Harbor Laboratory Press; Bovine EmbryoTransplantation (Ushi no Hai Ishoku), Hafez, Theriogenology (KachikuHanshokugaku), 5th edition, translation supervisors: S. Yoshida, J.Masaki, A. Iritani, Nishimura Shoten, 1992). Non-human animals to beproduced in the present invention include, for example, mice, rats,rabbits, guinea pigs, hamsters, dogs, cats, cows, horses, pigs, goats,and sheep, but are not particularly limited thereto.

It is possible to determine whether an sFLT1-encoding nucleic acid hasbeen introduced specifically into the trophectoderm of a blastocyst bythe methods of the present invention, by using methods known to thoseskilled in the art, for example, by amplifying introduced genes withPCR, or by detecting the expression of reporter genes such as EGFP orlacZ with a fluorescence or coloring method.

The present invention relates to methods for producing non-human mammalsthat show placenta-specific expression of sFLT1 including such steps.Furthermore, the present invention relates to methods for producingblastocysts in which an sFLT1-encoding nucleic acid is introducedspecifically into the trophectoderm.

The present invention also relates to a method of screening for asubstance that improves the symptoms of at least one disease selectedfrom the group consisting of pregnancy-induced hypertension syndrome,placental insufficiency, intrauterine growth retardation,glomerulosclerosis, and proteinuria, which comprises the steps of (a) to(c) below:

-   (a) obtaining a non-human mammal that shows placenta-specific    expression of sFLT1 described herein;-   (b) administering a test substance to the non-human mammal obtained    in step (a); and-   (c) selecting a substance that improves the symptoms of at least one    disease selected from the group consisting of pregnancy-induced    hypertension syndrome, placental insufficiency, intrauterine growth    retardation, glomerulosclerosis, and proteinuria, as compared to    when the test substance is not administered.

Substances obtained by screening methods of the present invention maybecome candidate compounds as therapeutic agents for at least onedisease selected from the group consisting of pregnancy-inducedhypertension syndrome, placental insufficiency, intrauterine growthretardation, glomerulosclerosis, and proteinuria. Therefore, “methods ofscreening for substances that improve the symptoms of at least onedisease selected from the group consisting of pregnancy-inducedhypertension syndrome, placental insufficiency, intrauterine growthretardation, glomerulosclerosis, and proteinuria” can also be describedas methods of screening for candidate compounds as therapeutic agentsfor at least one disease selected from the group consisting ofpregnancy-induced hypertension syndrome, placental insufficiency,intrauterine growth retardation, glomerulosclerosis, and proteinuria.

In the screening method of the present invention, first, a non-humanmammal that shows placenta-specific expression of sFLT1 described hereinis obtained. Such a non-human mammal can be obtained by theabove-described method.

Next, a test substance is administered to the non-human mammal thatshows placenta-specific sFLT1 expression of the present invention. Testsubstances include, for example, single compounds such as naturalcompounds, organic compounds, inorganic compounds, proteins, andpeptides, as well as compound libraries, expression products of genelibraries, cell extracts, cell culture supernatants, microbialfermentation products, marine organism extracts, and plant extracts, butare not limited thereto.

Administration of a test compound to a non-human mammal of the presentinvention that shows placenta-specific expression of sFLT1 can becarried out, for example, orally or parenterally, but is not limitedthereto. When a test compound is a protein, for example, a viral vectorcarrying a gene that encodes the protein is constructed, and the genecan be introduced into the genetically-modified non-human mammal of thepresent invention utilizing the infectivity of the viral vector.

In a screening method of the present invention, the last step is todetermine whether the symptoms of at least one disease selected from thegroup consisting of pregnancy-induced hypertension syndrome, placentalinsufficiency, intrauterine growth retardation, glomerulosclerosis, andproteinuria are improved by the test substance. Then, substances thatimprove the symptoms of at least one disease selected from the groupconsisting of pregnancy-induced hypertension syndrome, placentalinsufficiency, intrauterine growth retardation, glomerulosclerosis, andproteinuria, as compared to when the test substance is not administered,are selected. For example, when the level of PIGF mRNA in placenta orthe PIGF concentration in the mother's blood is increased, it isdetermined that the test substance improves the symptoms of at least onedisease selected from the group consisting of pregnancy-inducedhypertension syndrome, placental insufficiency, intrauterine growthretardation, glomerulosclerosis, and proteinuria. The level of PIGF mRNAcan be measured by Northern blotting, quantitative RT-PCR, microarrayanalysis, and such. Furthermore, the blood concentration can be measuredby Western blotting or ELISA.

Alternatively, blood pressure is measured or albumin/creatinine in urineis measured, and when these values decrease, the test substance isjudged to have improved symptoms of pregnancy-induced hypertensionsyndrome. Furthermore, it is judged that the test substance improvessymptoms in cases of glomerulosclerosis improvement when pathologicalsections of the kidney are observed, angioplasia improvement whenpathological sections of a placenta are observed, or recovery ofdecreased weight of the fetus and placenta.

Furthermore, the present invention relates to a pharmaceuticalcomposition (therapeutic agent) for treating at least one diseaseselected from the group consisting of pregnancy-induced hypertensionsyndrome, placental insufficiency, intrauterine growth retardation,glomerulosclerosis, and proteinuria, comprising a statin as an activeingredient. The present invention also provides a pharmaceuticalcomposition (therapeutic agent) for treating at least one diseaseselected from the group consisting of pregnancy-induced hypertensionsyndrome, placental insufficiency, intrauterine growth retardation,glomerulosclerosis, and proteinuria, comprising a placental growthfactor (PIGF) or a PIGF-encoding nucleic acid as an active ingredient.

The present inventors have confirmed that symptoms of pregnancy-inducedhypertension syndrome, placental insufficiency, intrauterine growthretardation, glomerulosclerosis, and proteinuria improve as a result ofadministering a statin, known as a therapeutic agent for hyperlipidemia,to the non-human model mammal developed by the present inventors.Furthermore, they confirmed that PIGF expression is induced by statinadministration. Accordingly, statins, PIGF, and PIGF-encoding nucleicacids (DNA, RNA) may be useful as therapeutic agents forpregnancy-induced hypertension syndrome, placental insufficiency,intrauterine growth retardation, glomerulosclerosis, and proteinuria.

Statins have the following effects in humans and non-human animals inwhich sFLT1 is specifically introduced into the placenta:

-   -   significantly reduces the concentration of sFLT1 in the mother's        blood;    -   does not affect the concentration of VEGF in the mother's blood;    -   significantly induces the level of PIGF mRNA and increases the        level by approximately five-fold particularly in the placenta;    -   significantly induces the concentration of PIGF in the mother's        blood;    -   ameliorates placental formation disorder;    -   ameliorates intrauterine growth restriction;    -   improves hypertension; and    -   improves renal dysfunction.

Furthermore, statins significantly increase circulating PIGF inwild-type pregnant female animals and non-pregnant animals. Furthermore,they do not lower blood pressure in normal pregnant female animals.

Pravastatin and atorvastatin induce PIGF production in human umbilicalvein endothelial cells (HUVEC).

Furthermore, PIGF has the following effects in non-human animals inwhich sFLT1 is introduced specifically into the placenta:

-   -   improves hypertension by impairing sFLT1 in the mother's blood;    -   improves renal dysfunction;    -   ameliorates placental formation disorder; and    -   ameliorates intrauterine growth restriction.

Statin (HMG-CoA reductase inhibitor) is a general term for substancesthat reduce the blood cholesterol level by inhibiting the function ofHMG-CoA reductase. Known statins include:

-   Mevastatin (synonym: compactin; chemical name:    [(1S,7S,8S,8aR)-8-[2-[(2R,4R)-4-hydroxy-6-oxooxan-2-yl]ethyl]-7-methyl-1,2,3,7,8,8a-hexahydronaphthalen-1-yl]    (2S)-2-methylbutanoate,    [(1S,7S,8S,8aR)-8-[2-[(2R,4R)-4-hydroxy-6-oxooxan-2-yl]ethyl]-7-methyl-1,2,4a,7,8,8a-hexahydronaphthalen-1-yl]    (2S)-2-methylbutanoate,    [8-[2-(4-hydroxy-6-oxooxan-2-yl)ethyl]-7-methyl-1,2,3,7,8,8a-hexahydronaphthalen-1-yl]    2-methylbutanoate, etc.);-   Atorvastatin (chemical name: (−)-Monocalcium bis    {(3R,5R)-7-[2-(4-fluorophenyl-5-isopropyl-3-phenyl-4-phenylcarbamoyl-1H-pyrrol-1-yl]-3,5-dihydroxyheptanoate}trihydrate,    etc.);-   Simvastatin (chemical name:    (1S,3R,7S,8S,8aR)-8-{2-[(2R,4R)-4-Hydroxy-6-oxotetrahydro-2H-pyran-2-yl]-ethyl}-3,7-dimethyl-1,2,3,7,8,8a-hexahydronaphthalen-1-yl    2,2-dimethylbutanoate, etc.);-   Cerivastatin (chemical name:    (E,3R,5S)-7-[4-(4-fluorophenyl)-5-(methoxymethyl)-2,6-di(propan-2-yl)pyridin-3-yl]-3,5-dihydroxyhept-6-enoic    acid, etc.);-   Pitavastatin (chemical name: (+)-monocalcium bis    {(3R,5S,6E)-7-[2-cyclopropyl-4-(4-fluorophenyl)-3-quinolyl]-3,5-dihydroxy-6-heptenoate},    etc.);-   Pravastatin (chemical name:    Monosodium(3R,5R)-3,5-dihydroxy-7-{(1S,2S,6S,8S,8aR)-6-hydroxy-2-methyl-8-[(2S)-2-methylbutanoyloxy]-1,2,6,7,8,8a-hexahydronaphthalen-1-yl}heptanoate);-   Fluvastatin (chemical name:    (±)-(3RS,5SR,6E)-Sodium-7-[3-(4-fluorophenyl)-1-(1-methylethyl)-1H-indol-2-yl]-3,5-dihydroxy-6-heptenoate,    etc.);-   Rosuvastatin (chemical name: Monocalcium    bis((3R,5S,6E)-7-{4-(4-fluorophenyl)-6-isopropyl-2-[methanesulfonyl(methyl)amino]pyrimidin-5-yl}-3,5-dihydroxyhept-6-enoate),    etc.); and-   Lovastatin (chemical name:    [(1S,3R,7S,8S,8aR)-8-[2-[(2R,4R)-4-hydroxy-6-oxooxan-2-yl]ethyl]-3,7-dimethyl-1,2,3,7,8,8a-hexahydronaphthalen-1-yl]    (2S)-2-methylbutanoate), etc.).

Those skilled in the art can synthesize statins besides mevastatinaccording to the description in Japanese Patent Kohyo Publication No.(JP-A) 2009-538831 (unexamined Japanese national phase publicationcorresponding to a non-Japanese international publication). Furthermore,those skilled in the art can obtain mevastatin, for example, by themethod described in the Journal of Lipid Research, Volume 33, 1992.Alternatively, pravastatin and atorvastatin can be purchased from CaymanChemical and Toronto Research Chemicals, respectively. Furthermore, theycan be obtained from many pharmaceutical companies as prescriptiondrugs.

The PIGF genes and proteins encoded by these genes are known. Forexample, a mouse PIGF gene and a protein encoded by the gene are knownas GenBank: BC016567.1, and a human PIGF gene and a protein encoded bythe gene are known as GenBank: BC001422.2. The nucleotide sequence ofthe mouse PIGF gene is shown in SEQ ID NO: 3, and the protein encoded bythe gene is shown in SEQ ID NO: 4. The nucleotide sequence of the humanPIGF gene is shown in SEQ ID NO: 5, and the protein encoded by the geneis shown in SEQ ID NO: 6.

PIGF genes and proteins encoded by these genes are also known forchimpanzees, rats, dogs, cows, horses, pigs, sheep, and such. Therespective accession numbers are shown below.

NCBI Reference Sequence chimpanzee gene XM_001158223.1 proteinXP_001158223.1 rat gene NM_053595.2 protein NP_446047.1 dog geneXM_849639.1 protein XP_854732.1 cow gene NM_173950.2 protein NP_776375.1horse gene XM_001491442.1 protein XP_001491492.1 Genbank pig geneFJ177137.1 protein ACI24003.1 sheep gene AY157708.1 protein AAN77495.1

“PIGF-encoding nucleic acids” in the pharmaceutical agents of thepresent invention include “PIGF-encoding DNA” and “PIGF-encoding RNA”.The form of “PIGF-encoding DNA” is not particularly limited, and may bea genomic DNA, cDNA, synthetic DNA, or vector containing such DNA.

Furthermore, in addition to as a naturally-occurring protein, “PIGF” ina pharmaceutical composition (therapeutic agent) of the presentinvention can be prepared as a recombinant protein using known geneticengineering techniques. In addition, the organism from which “PIGF” in apharmaceutical composition (therapeutic agent) of the present inventionis derived is not particularly limited. When it is used for treatment orprevention of human diseases, it is preferably derived from a mammal,and most preferably derived from a human. The naturally-occurringprotein can be prepared, for example, by affinity chromatography methodsthat utilize antibodies against PIGF in tissue extracts such as theplacenta which is believed to express PIGF.

On the other hand, recombinant proteins can be prepared, for example, asa recombinant polypeptide by methods known to those skilled in the art.Recombinant polypeptides can be prepared, for example, by inserting aPIGF-encoding nucleic acid into a suitable expression vector, collectingtransformants obtained by introducing this vector into suitable hostcells, and after obtaining an extract thereof, purifying it bychromatography such as ion exchange, reverse phase, or gel filtration,or by affinity chromatography in which antibodies against PIGF areimmobilized onto the column, or by combining a plurality of suchcolumns.

Alternatively, when PIGF is expressed as a fusion polypeptide with aglutathione S-transferase protein, or as a recombinant polypeptide withmultiple additions of histidines in host cells (for example, an animalcell or Escherichia coli), the expressed recombinant polypeptide can bepurified using a glutathione column or a nickel column.

With regard to the above vectors, for example, when the host is E. coli,as long as the vector has an “ori” for amplification in E. coli suchthat the vector is amplified and prepared in large quantities in E. coli(for example, JM109, DH5α, HB101, and XL1Blue) or such, and further hasa selection gene for transformed E. coli (for example, a drug resistancegene that allows differentiation using a certain drug (ampicillin,tetracycline, kanamycin, or chloramphenicol)), the vectors are notparticularly limited. The vectors include, for example, M13 vectors, pUCvectors, pBR322, pBluescript, and pCR-Script. In addition to the abovevectors, for example, pGEM-T, pDIRECT, and pT7 can also be used for thesubcloning and excision of cDNAs. When using vectors to produce PIGF,expression vectors are particularly useful. When an expression vector isexpressed in E. coli, for example, it should have the abovecharacteristics in order to be amplified in E. coli. Additionally, whenE. coli such as JM109, DH5α, HB101, or XL1-Blue are used as the host,the vector must have a promoter that allows efficient expression in E.coli, for example, a lacZ promoter (Ward et al. Nature 341: 544-546,1989; FASEB J. 6: 2422-2427, 1992), araB promoter (Better et al. Science240: 1041-1043, 1988), or T7 promoter. Other examples of the vectorsinclude pGEX-5X-1 (Pharmacia), “QIAexpress system” (QIAGEN), pEGFP, andpET.

Furthermore, the vector may comprise a signal sequence for polypeptidesecretion. When producing polypeptides into the periplasm of E. coli,the pelB signal sequence (Lei, S. P. et al. J. Bacteriol. 169: 4379(1987)) may be used as a signal sequence for polypeptide secretion. Forexample, calcium chloride methods or electroporation methods may be usedto introduce the vector into a host cell.

In addition to E. coli, expression vectors derived from mammals (e.g.,pcDNA3 (Invitrogen), pEGF-BOS (Nucleic Acids Res. 18(17): 5322 (1990)),pEF, and pCDM8), insect cells (e.g., “Bac-to-BAC baculovirus expressionsystem” (GIBCO-BRL) and pBacPAK8), plants (e.g., pMH1 and pMH2), animalviruses (e.g., pHSV, pMV, and pAdexLcw), retroviruses (e.g., pZIPneo),yeasts (e.g., “Pichia Expression Kit” (Invitrogen), pNV11 and SP-Q01),and Bacillus subtilis (e.g., pPL608 and pKTH50) may also be used asvectors for producing PIGF.

For expression in animal cells such as CHO, COS, and NIH3T3 cells, thevector must have a promoter necessary for expression in such cells, forexample, an SV40 promoter (Mulligan et ai. Nature 277: 108 (1979)),MMLV-LTR promoter, EF1α promoter (Mizushima et ai. Nucleic Acids Res.18: 5322 (1990)), or CMV promoter. It is even more preferable that thevector comprises a gene for selecting transformants (for example, adrug-resistance gene enabling differentiation by a drug (such asneomycin and G418)). Examples of vectors with such characteristicsinclude pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

On the other hand, in vivo polypeptide production systems include thoseusing animals or plants. A PIGF-encoding nucleic acid is introduced intothe animals or plants to produce PIGF in the body of the animals orplants, and polypeptides are collected from them.

Production systems using animals include those that use mammals orinsects. Mammals that can be used include goats, pigs, sheep, mice, cowsand such (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). Whenmammals are used, transgenic animals can be used.

For example, nucleic acid encoding PIGF can be prepared as a fusion genewith a gene encoding a polypeptide such as goat β casein which isuniquely produced into milk. Next, DNA fragments comprising the fusiongene are injected into goat embryos, and the embryos are introduced intofemale goats. PIGF can be obtained from milk produced by the transgenicanimals born to the goats that received the embryos, or produced fromprogenies of these animals. The transgenic goats can be given hormonesto increase the volume of milk containing the polypeptide that theyproduce (Ebert, K. M. et al., Bio/Technology (1994) 12, 699-702).

As insects, for example, silkworms can be used. When silkworms are used,PIGF is obtained from the body fluids of silkworms (Susumu, M. et al.,Nature (1985) 315, 592-594) after the silkworms are infected with abaculovirus into which a nucleic acid encoding PIGF has been inserted.

Moreover, when plant is used, for example, tobacco can be used. Whentobacco is used, a nucleic acid encoding PIGF is inserted into a plantexpression vector (e.g., pMON 530) and the vector is introduced intobacteria such as Agrobacterium tumefaciens. This bacterium is used toinfect tobacco (e.g., Nicotiana tabacum) such that PIGF can be obtainedfrom the leaves of this tobacco (Julian K.-C. Ma et al., Eur. J.Immunol. (1994) 24, 131-138).

The resulting PIGF may be isolated from the inside or outside (such asmedium) of host cells, and purified as substantially pure and homogenouspolypeptides. Methods for isolation and purification commonly used forpolypeptide purification may be used for the isolation and purificationof polypeptides, and they are not limited to any method. Polypeptidesmay be isolated and purified by appropriately selecting and combining,for example, column chromatographies, filtration, ultrafiltration,salting out, solvent precipitation, solvent extraction, distillation,immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectricfocusing, dialysis, and recrystallization.

By treating PIGF of the present invention with a suitable proteinmodification enzyme before or after purification, PIGF can be optionallymodified and peptides can be removed partially. For example, trypsin,chymotrypsin, lysyl endopeptidase, protein kinase, glucosidase, and suchare used as protein modification enzymes.

Subjects to be administered with pharmaceutical compositions(therapeutic agents) of the present invention are mammals. Mammalsinclude, but are not limited to, humans, mice, rats, rabbits, guineapigs, hamsters, dogs, cats, cows, horses, pigs, goats, and sheep.

The above-mentioned therapeutic agents can be administered orally orparenterally, but parenteral administration is preferred. Specificexamples include injections, transnasal administrations, transpulmonaryadministrations, and transdermal administrations. For example,injections can be administered systemically or locally by intravenousinjection, intramuscular injection, intraperitoneal injection, orsubcutaneous injection.

When administering PIGF-encoding nucleic acids to a living body, viralvectors such as retrovirus, adenovirus, and Sendai virus, and non-viralvectors such as liposomes may be used. Examples of administrationmethods include in vivo methods and ex vivo methods.

A therapeutic agent of the present invention can be directlyadministered to patients alone, and alternatively it can be administeredas a formulated pharmaceutical agent by known pharmaceutical methods.For example, it can be used in the form of an injection of a sterilesolution or suspension with water or other pharmaceutically acceptableliquids. Furthermore, for example, it may be formulated by appropriatelycombining with pharmacologically acceptable carriers or vehicles,specifically sterilized water and physiological saline, emulsifiers,suspending agents, surfactants, stabilizers, vehicles, antiseptics, andsuch, and mixed in a unit dosage form required by generally acceptedpharmaceutical procedures. The amount of active ingredient in theseformulations is adjusted so that one can obtain an appropriate amount inthe specified range.

Sterile compositions for injection can be formulated using vehicles suchas distilled water for injection, according to standard protocols.Aqueous solutions used for injection include, for example, physiologicalsaline and isotonic solutions containing glucose or other adjuvants suchas D-sorbitol, D-mannose, D-mannitol, and sodium chloride. These can beused in conjunction with suitable solubilizers such as alcohol,specifically ethanol, polyalcohols such as propylene glycol andpolyethylene glycol, and non-ionic surfactants such as Polysorbate 80™and HCO-50.

Oils include sesame oils and soybean oils, and can be combined withsolubilizers such as benzyl benzoate or benzyl alcohol. These may alsobe formulated with buffers, for example, phosphate buffers or sodiumacetate buffers; analgesics, for example, procaine hydrochloride;stabilizers, for example, benzyl alcohol or phenol; or antioxidants. Theprepared injections are typically aliquoted into appropriate ampules.

Furthermore, the administration dose can be appropriately selectedaccording to the age and symptoms of the patient. The dose can beselected, for example, from the range of 0.0001 to 1,000 mg per kg ofbody weight. Alternatively, the dose may be, for example, in the rangeof 0.001 to 100,000 mg/patient. However, the pharmaceutical agents ofthe present invention are not limited to these doses.

Daily administration from the early stage of hypertension is preferredfor the therapeutic agents of the present invention, particularlypravastatin. More specifically, in the case of humans, for example,daily administration from the 20th week of pregnancy or earlier ispreferred. For example, in the case of mice, daily administrationpreferably from day 13, more preferably from day 10, and even morepreferably from day 7 is preferred.

Furthermore, it is more preferable that daily administration of atherapeutic agent of the present invention is started before the onsetof hypertension.

All prior art references cited herein are incorporated by reference intothis description.

EXAMPLES

Herein below, the present invention will be specifically describedfurther using the Examples, but the technical scope of the presentinvention is not to be construed as being limited thereto.

1. Method

Primers and PCR

The human sFLT1 (hsFLT1) cDNA was amplified by RT-PCR from HUVECs(Kurabo) with primers 5′-aaggatccgccgccatggtcagctactgggac-3′ (SEQ ID NO:7) and 5′-ttctcgagttaatgttttacattactttgtgtg-3′ (SEQ ID NO: 8). The mousePIGF (mPIGF) cDNA was amplified by RT-PCR from E13.5 placenta withprimers 5′-aagaattcgccaccatgctggtcatgaagctgttc-3′ (SEQ ID NO: 9) and5′-ttctcgagtcacgggtggggttcctcag-3′ (SEQ ID NO: 10). In FIG. 1b , primers5′-aagtgtgacgttgacatccg-3′ (SEQ ID NO: 11) and5′-gatccacatctgctggaagg-3′ (SEQ ID NO: 12) were used for amplificationof mActb, and primers 5′-ggctgagcataactaaatctgcc-3′ (SEQ ID NO: 13) and5′-ggaatgacgagctcccttccttca-3′ (SEQ ID NO: 14) were used foramplification of hsFLT1. In FIG. 2c , primers5′-catccgtaaagacctctatgccaac-3′ (SEQ ID NO: 15) and5′-atggagccaccgatccaca-3′ (SEQ ID NO: 16) were used for amplification ofmActb, and primers 5′-tgctgggaacaactcaacag-3′ (SEQ ID NO: 17) and5′-cctcatcagggtattcatcca-3′ (SEQ ID NO: 18) were used for amplificationof mPIGF.

Lentiviral Vectors

The HIV-I based, self-inactivating, lentiviral vector plasmid pLV-EGFPwas described previously (Nat Biotechnol. 2007 February; 25(2): 233-7).Other lentiviral vector plasmids pLV-hsFLT1 and pLV-mPGF were preparedby substituting an EGFP cDNA with an hsFLT1 cDNA or an mPIGF cDNA,respectively. Vesicular stomatitis virus glycoprotein-pseudotypedlentiviral vectors were generated, and the p24 gag antigen concentrationwas measured as described previously (Nat Genet. 2000 June; 25(2):217-22).

Mice and Lentiviral Transduction

Wild-type B6D2F1 female animals were superovulated by human chorionicgonadotropin (5 units) 48 hours after the intraperitoneal injection ofpregnant mare's serum gonadotropin (5 units) and then mated withwild-type B6D2 F1 male animals. Two to four cell-stage embryos werecollected from the female animals at 1.5 days after copulation, and thenincubated in the kSOM medium (erbach BOR 1994) for two days to obtainblastocysts. Zona pellucida was removed in acidic Tyrode's solution(Sigma, Nicolson JCB 1975) to prepare zona pellucida-free blastocysts.Then, they were incubated individually for 4 hours in 5 μL of mediumcontaining lentiviral vectors. The transduced blastocysts were washedthree times and then transplanted into pseudo-pregnant ICR femaleanimals. The present inventors transplanted ten blastocysts into eachhorn of the uterus. All animal experiments were approved by the AnimalCare and Use Committee of the Research Institute for Microbial Diseases,Osaka University.

Statins

Pravastatin sodium salt (Cayman Chemical) was dissolved in 100% ethanolto produce a stock solution (1 mg/mL), which was diluted by sterile PBS(25 μg/mL) before use and intraperitoneally injected every day startingfrom E7.5 to E18.5 at 5 μg/animal unless specifically instructedotherwise.

Atorvastatin (Toronto Research Chemicals Inc.) was dissolved in 100%methanol to produce a stock solution (25 mg/mL), which was diluted bysterile PBS (12.5 μg/mL) before use.

For HUVEC cells, each of the stock solutions was added upon dilution to1 μM or 10 μM in a culture medium.

Blood and Urinary Samples

Blood samples were allowed to clot and were centrifuged to prepare serumsamples. Concentrations of hsF1T1, mVEGF, and mPIGF were measured withELISA kits, according to the manufacturer's instructions (R&D system).Aspartate aminotransferase (AST) and alanine transaminase (ALT) weremeasured by Fuji dry-chem 3500V and dri-chem slides (Fuji Film.co).Urine samples were collected at E18.5. Urine albumin and creatinineconcentrations were measured by using the Fuji dry-chem 3500V anddri-chem slides (Fuji Film.co, Tokyo Japan).

Measurement of Blood Pressure

Blood pressure was measured by the tail-cuff method (Softron Ltd, Tokyo,Japan). The mice were lightly fixed in a small cage without anesthesia,and their blood pressure was measured after their behavior, heart rate,and blood pressure stabilized. After stabilization, both systolic anddiastolic blood pressures were recorded at least five times until thestabilization became unstable. Mean values of both the systolic anddiastolic blood pressures measured as mentioned above were used forfurther statistical analysis.

Histopathology of Placenta

Placentas collected from sacrificed mice were fixed in 4%paraformaldehyde (PFA)/PBS for 12 hours, and then subjected to furtherstaining. Anti-CD31 antibody staining has been described previously. Inbrief, the PFA-fixed samples were rinsed with PBS for 4 hours, andsoaked sequentially in 40%, 70%, and 100% methanol at 4° C. Sectionsprepared at a thickness of 5 μm were stained with an anti-mouse CD31antibody (BD Biosciences) and visualized with an AlexaFluor488-conjugated goat anti-rat IgG (Molecular Probes). Morphologicalalterations of the tissues were analyzed by Ehrlich hematoxylin-eosinstaining. Samples were visualized by using conventional microscopy(DM5500 B; Leica), and images were processed using the Adobe PhotoshopCS3 software (Adobe Systems).

In vitro Expression of PIGF as an Effect of Statin

HUVEC and HEK293T cells were plated at 2×10⁴ and 1×10⁵ cells per well in6-well plates, respectively, and incubated under 5% CO₂ at 37° C. After24 hours, the medium was replaced with a fresh medium containing or notcontaining a statin. The media collected after another 24 hours werecentrifuged at 1,000×g, and the supernatants were subjected to ELISA.

Statistical Analyses

All values are expressed as mean±s.e.m. The present inventors used thetwo-tailed unpaired Student's t-test for comparison between groups, andP values<0.05 were considered as significant.

2. Results

hsFLT1 was expressed specifically in the murine placenta throughout thegestational period to develop a novel animal model for pregnancy-inducedhypertension syndrome (FIG. 1). The present inventors previously showedthat when the trophectoderm cells of blastocysts are transduced with alentiviral (LV) vector, placenta-specific gene integration andexpression take place (Nat. Biotechnol. 2007 February; 25 (2): 233-7).According to this method, the present inventors transduced zonapellucida-free blastocysts with an hsFLT1-expressing LV vector(LV-hsFLT1), and transplanted these blastocysts into pseudo-pregnantfemale animals (FIG. 1a ). Unless specifically indicated otherwise,blastocysts were transduced with the lentiviral vector at 100 ng-p24/mLthroughout the experiment. As predicted by the present inventors, PCRand RT-PCR analyses demonstrated that integration and expression of theLV-hsFLT1 transgene was placenta-specific (FIG. 1b ), butplacenta-derived hsFLT1 was proven to circulate in the mother's blood(FIGS. 1c and d ).

The hsFLT1 concentration in the mother's blood correlated with theamount of lentiviral vector at the time of blastocyst transduction (FIG.1c ). When LV-hsFLT1 was transduced at 100 ng-p24/mL, the circulatinghsFLT1 concentration in the mother's body gradually increased duringpregnancy, reaching an average value of 5.84±1.26 ng/mL at the embryonicage of 18.5 days (E18.5) (FIG. 1d ), which was comparable to the sFLT1concentration (4,382 pg/mL on average) in human patients (N Engl J Med.2004 Feb. 12; 350(7): 672-83., Circulating angiogenic factors and therisk of preeclampsia.). After the elevation of hsFLT1, the systolic aswell as diastolic blood pressures significantly increased at E16.5 andcontinued during the rest of pregnancy (P<0.05, FIG. 1e ). It should benoted that the blood pressure promptly reached a normal levelpostpartum, which mimics recovery after delivery of the placenta inhuman. The symptoms of hypertension were also observed in the grouptreated with LV-hsFLT1 at 20 ng-p24/mL, but not in the group treated at4 ng-p24/mL (FIG. 1e ). The pregnant female animals carrying theLV-hsFLT1-transduced placenta exhibited glomerulosclerosis (FIG. 4) andproteinuria (FIG. 1f ). These data indicated that the placenta-specificoverexpression of hsFLT1 provides an animal model for pregnancy-inducedhypertension syndrome.

Next, the present inventors used their model mice to examine thetherapeutic effects of a HMG-CoA reductase inhibitor, pravastatin, onpregnancy-induced hypertension syndrome. Statins are generally used asdrugs for hypercholesterolemia, but it has been recently reported thatstatins have a protective effect on vascular endothelial cells(Pharmacol Ther. 2009 April; 122(1): 30-43. Epub 2009 Jan. 23, Statinsand cardioprotection—more than just lipid lowering?; and Circulation.2002 Feb. 12; 105(6): 739-45., Statins have biphasic effects onangiogenesis). Moreover, pravastatin administration improved placentaldysfunction and prevented miscarriages in a spontaneous-abortion modelmouse (Blood. 2009 Apr. 23; 113(17): 4101-9. Epub 2009 Feb. 20,Pravastatin prevents miscarriages in mice: role of tissue factor inplacental and fetal injury). To define its prophylactic/therapeuticeffect, the present inventors intraperitoneally administered pravastatinat 5 μg/head, which is equivalent to a therapeutic dose of 10 mg/60 kgin human and the like. When pravastatin was administered every day fromE10.5 or earlier, a prophylactic/therapeutic effect on hypertension wasobserved (FIG. 2a ). Administration from E13.5 also similarly decreasedthe blood pressure, but the decrease was not significant. It should benoted that pravastatin is not hypotensive in normal pregnant femaleanimals (FIG. 5). Glomerulosclerosis and proteinuria were similarlyimproved in the treated mice (FIG. 4 and Table 1). The P values in Table1 are all t-test values in comparison to the control.

TABLE 1 ALBUMIN/CREATININE LV n AVERAGE P LV-GFP 12 5.27 ± 0.74 —LV-hsFLT1 14 7.47 ± 0.61 0.03 LV-hsFLT1 + PRAVASTATIN 8 4.06 ± 0.38 0.23LV-hsFLT1 + LV-mPIGF 15 6.41 ± 0.61 0.24 Average ± S.E.M.

In the next experiment, the present inventors investigated howpravastatin improved sFLT1-induced hypertension. Since sFLT1 interactswith and antagonizes the angiogenic functions of VEGF and PIGF, thepresent inventors measured the placental mRNA levels and the maternalblood concentrations of these factors at E18.5 (FIGS. 2b and c ).Pravastatin was administered every day from E7.5. Pravastatin did notaffect the hsFLT1 mRNA level, but the circulating hsFLT1 wassignificantly decreased from 5.84±1.26 to 0.99±0.65 ng/mL (n=5, P<0.001)in the female animals carrying LV-hsFLT1-transduced placentas. Theresulting hsFLT1 concentration was equal to or lower than theconcentration in pregnant women with normal blood pressure (1,642 ng/mLon average; N Engl J Med. 2004 Feb. 12; 350(7): 672-83. Circulatingangiogenic factors and the risk of preeclampsia). mVEGF was not changedin terms of mRNA and protein levels (from 44.7±22.3 to 35.5±13.6 pg/mL,n=6, P=0.36). Interestingly, mPIGF was significantly induced by thepravastatin treatment (60.3±12.6 to 116.6±13.2 pg/ml; n=7; P<0.001).Accordingly, placental mPIGF mRNA increased approximately five-foldafter the pravastatin treatment (FIG. 2c ).

To determine whether pravastatin-induced PIGF counteracts sFLT1 in vivo,the present inventors simultaneously expressed mPIGF with hsFLT1 in theplacenta by transducing the blastocyst with LV-hsFLT1 and LV-mPIGF(FIGS. 2a and b ). As predicted, overexpression of mPIGF decreasedhsFLT1 in the mother's blood (FIG. 2b ), and improved hypertension (FIG.2a ). LV-mPIGF transduction alone did not show any hypotensive effects(FIG. 5). In addition to hypertension, glomerulosclerosis andproteinuria were similarly improved by mPIGF (Table 1 and FIG. 4). Thesedata support the idea that pravastatin-induced PIGF counteracts sFLT1and improves the symptoms of pregnancy-induced hypertension syndrome invivo.

To determine whether pravastatin induces PIGF without influences fromthe LV vector or sFLT1, the present inventors administered pravastatinto wild-type female animals. The circulating mPIGF was significantlyinduced in the pregnant female animals from 52.4±20.2 pg/mL to152.0±19.7 pg/mL (n=4, P<0.001, FIG. 3a ). Surprisingly, mPIGF was alsoinduced in the non-pregnant female animals from 0 pg/mL to 29.8±13.2pg/mL (n=5, P<0.01, FIG. 3a ). As far as the present inventors know,this is the first report that a therapeutic dose of pravastatin inducesPIGF expression in vivo. Next, the present inventors examined whetherpravastatin induces PIGF production in the vascular endothelial cells.Both pravastatin and atorvastatin induced PGF production in humanumbilical vein endothelial cells (HUVECs). The statin treatment did notproduce PIGF in the control human embryonic kidney (HEK293) cells (FIG.3b ). Statin has been reported to be effective for vascular dysfunction,but its mechanism is still unclear (Pharmacol Ther. 2009 April; 122(1):30-43. Epub 2009 Jan. 23, Statins and cardioprotection—more than justlipid lowering?). The data obtained by the present inventors suggestedthat the ectopic expression of PIGF may explain the protective effect ofstatins on vascular endothelial cell dysfunction.

Finally, taking advantage of the present inventor's placenta-specificgene manipulation method, the present inventors evaluated the localeffects of excess sFLT1 on placental formation and fetal development.When examined at E13.5, immunostaining of PECAM1 using an anti-CD31antibody showed suppression of vascular bed development in theLV-hsFLT1-transduced placenta (FIG. 3a ). When Caesarean section wasperformed at E18.5, both placenta and fetus from the LV-hsFLT1-treatedfemale animals were smaller than those from the control LV-EGFP-treatedgroup (FIGS. 3b to d ), but the implantation rate and birth rate wereequivalent (Table 2). The impaired placental formation and IUGR wereameliorated by the pravastatin treatment and the placenta-specific mPIGFexpression (FIG. 3). These data support the idea that statin-inducedPIGF counteracts sFLT1 and ameliorates impaired placental formation andIUGR in the pregnancy-induced hypertension syndrome models of thepresent inventors.

TABLE 2 IMPLANTATION RATE AND BIRTH RATE GENE IMPLANTATION BIRTH LV nTRANSFER RATE (%) RATE (%) LV-GFP 11 220  99 82 (45.0 ± 1.8) (37.3 ±1.7) LV-hsFLT1 11 220 100 85 (45.5 ± 2.0) (38.6 ± 2.5) LV-hsFLT1 + 10200  89 76 PRAVASTATIN (44.5 ± 0.9) (38.0 ± 1.0) LV-hsFLT1 + 12 240 10890 LV-mPIGF (45.0 ± 1.2) (37.5 ± 1.3) Average ± S.E.M.

The experiments conducted so far showed that VEGF improves hypertension(Hypertension. 2007 October; 50(4): 686-92. Epub 2007 Aug. 27,Recombinant vascular endothelial growth factor 121 attenuateshypertension and improves kidney damage in a rat model of preeclampsia).However, in the present inventors' system, the therapeutic effect ofpravastatin depends on PIGF rather than on VEGF. This idea is alsosupported by the fact that recombinant PIGF improves hypertensioninduced by sFLT1 which was expressed by an adenovirus (Hypertension.2009 November; 54(5): 1129-35. Epub 2009 Sep. 28, Effect of recombinantplacental growth factor 2 on hypertension induced by full-length mousesoluble fms-like tyrosine kinase 1 adenoviral vector in pregnant mice).While the VEGFRI-mediated vasodilatory effect of PIGF cannot be excluded(Am J Physiol Heart Circ Physiol. 2008 March; 294(3): H1381-7. Epub 2008Jan. 11, Placental growth factor is a potent vasodilator of rat andhuman resistance arteries), the present inventors' data suggested thatthe direct antagonizing effect of PIGF on sFLT1 is an important elementduring pravastatin treatment.

Regarding clinical applications of statins, teratogenicity is a majorconcern for pregnant women. The FDA classifies statins as category X andstrongly discourages the prescription of statins during the firsttrimester. However, several recent examinations indicate that statinsmay be safe even in the first trimester (Reprod Toxicol. 2008 October;26(2): 175-7. Epub 2008 Jul. 1, Prenatal exposure to HMG-CoA reductaseinhibitors: effects on fetal and neonatal outcomes., Br J ClinPharmacol. 2007 October; 64(4): 496-509. Epub 2007 May 15, Risk ofcongenital anomalies in pregnant users of statin drugs., Pregnancyoutcomes after maternal exposure to simvastatin and lovastatin., ReprodToxicol. 1996 November-December; 10(6): 439-46., Postmarketingsurveillance of lovastatin and simvastatin exposure during pregnancy).The present inventors also did not notice any deformity in the pups ofthe pravastatin-treated female animals used in the experiments. Whilelarge-scale and accurate morphological examinations are necessary,statins are highly promising candidate substances forprevention/improvement of pregnancy-induced hypertension syndrome, inorder to save numerous pregnant women and infants from morbidity andmortality worldwide.

INDUSTRIAL APPLICABILITY

Model animals for pregnancy-induced hypertension syndrome that showplacenta-specific expression of sFLT1, and methods for producing themodel animals are provided by the present invention. Conventional modelanimals for pregnancy-induced hypertension syndrome have been producedby overexpressing a causative factor in the mother's body, and they aredefective as disease models. On the other hand, in the model animals ofthe present invention, symptoms of hypertension appear along withplacental growth as pregnancy continues, and symptoms of hypertensionare improved by placental shedding at delivery.

Specifically, the model animals of the present invention have thefollowing advantages compared to conventional model animals.

-   (1) Use of a lentiviral vector in a preferred embodiment leads to    not transient but constitutive sFLT1 expression throughout the    pregnancy period.-   (2) While sFLT1 is conventionally expressed mainly in the liver by    gene transfer into the mother's body, the method of the present    invention expresses sFLT1 by introducing the gene transfer only into    the placenta without gene transfer into the mother's body. This    eliminates direct effects of viral vector infection on the mother's    body, which has been conventionally impossible. Furthermore, it can    exclude direct effects of sFLT1 gene expression in the mother's    body.-   (3) In the conventional method, since sFLT1 is expressed throughout    the mother's body, the pathological condition is maintained    regardless of delivery, whereas in the method of the present    invention, the expression is placenta-specific; and thus the    pathological condition is improved after delivery. Since humans    follow the same course, one can say that this model better reflects    the patient's pathological condition.

Furthermore, in the present invention, statins were found to improve thesymptoms of pregnancy-induced hypertension syndrome, placentalinsufficiency, intrauterine growth retardation, glomerulosclerosis, andproteinuria. At the moment, the only existing methods for treatingpregnancy-induced hypertension syndrome are delivery or removal of theplacenta by Caesarian section. The economic effect is great with theadded indication for statins as therapeutic agents for pregnancy-inducedhypertension syndrome, affecting 5 to 7% of all pregnant women.Furthermore, the economic effect is also great because the burden on theobstetrical department caused by emergency Caesarian section, as well asthe burden on neonatal ICU due to early Caesarian section and such canbe lessened.

The invention claimed is:
 1. A mouse pregnant with a mouse fetus,comprising: a placenta comprised of cells differentiated fromtransplanted cells transfected in vitro with a viral vector comprised ofa nucleic acid sequence encoding human soluble fms-like tyrosine kinase1 (sFLT1), the nucleic acid sequence operatively inserted in the viralvector in a manner such that the human sFLT1 protein is specificallyexpressed in the mouse placenta, wherein the pregnant mouse exhibitssymptoms of a disease selected from the group consisting ofpregnancy-induced hypertension syndrome, placental insufficiency,intrauterine growth retardation, glomerulosclerosis, and proteinuria asa result of the expression of the human sFLT1 protein in the mouseplacenta; and wherein the human sFLT1protein is expressed in the mouseplacenta throughout a gestational period of the mouse fetus.
 2. Thepregnant mouse of claim 1, produced by: (a) removing zona pellucida of ablastocyst of a mouse; (b) introducing a viral vector comprising anucleic mouse acid sequence encoding human sFLT1-encoding nucleic acidspecifically into trophectoderm of the blastocyst obtained in step (a);and (c) transplanting the blastocyst obtained in step (b) into asuperovulated female mouse.
 3. The pregnant mouse of claim 1, whereinthe viral vector is selected from the group consisting of a retroviralvector and adenovirus vector.
 4. The pregnant mouse of claim 2, whereinthe viral vector is selected from the group consisting of a retroviralvector and adenovirus vector.
 5. The pregnant mouse of claim 3, whereinthe superovulated female mouse is a Wild-Type B6D2Fl female mouse. 6.The pregnant mouse of claim 4, wherein the superovulated female mouse isa Wild-Type B6D2Fl female mouse.
 7. The pregnant mouse of claim 5,wherein the human sFLT1-encoding nucleic acid sequence comprises SEQ IDNO:
 1. 8. The pregnant mouse of claim 6, wherein the humansFLT1-encoding nucleic acid sequence comprises SEQ ID NO:
 1. 9. Thepregnant mouse of claim 7, wherein the mouse exhibits symptoms ofpregnancy-induced hypertension.
 10. The pregnant mouse of claim 8,wherein the mouse exhibits symptoms of pregnancy-induced hypertension.